Possible Items to Do for this project: -- Dr. B
IC50 determination of top compounds
DSF analysis of ligands (machine in Robertus lab) DSF_NatProt2007.pdf
pharmacophore searching in ICM http://www.molsoft.com/gui/cheminformatics-tutorials.html#tutorial-3D-pharmacophore
Ensemble docking in ICM and GOLD
-- i.e. docking against multiple receptor conformations. How many PDB structures are there for your enzyme?
In silico alteration of one amino acid residue that is most likely to be mutated in a resistant form of the bacterium and then virtual screening against that structure with your same top hits
-- this mutant version could then be grown up and test in wet lab against the same compounds
Molecular dynamics simulations and re-scoring of top ligands
crystallization
WITH L(+)-tartrate ion Temperature: 293K Final Optimized condition: 10%(v/v) Tascimate, 0.1 M HEPES pH 7.0 and 19%(w/v) PEG 3350 Extra Info:
Equal volumes of the protein (1.5 ml) and the reservoir
(1.5 ml) were mixed and allowed to equilibrate with 1.0 ml of reservoir
for 24–48 h. NOTE: Tacsimate (Hampton Research, HR2-755) is a mixture of organic acids that includes sodium malonate, sodium acetate, triammonium citrate, succinic acid, dl-malic acid and sodium formate
Inhibitor Free:
These crystals were grown in sitting drops at room temperature using a protein stock solution of 10 mg/mL enzyme in 50 mM sodium acetate pH 6.0, and reservoir solutions consisting of 0.05 – 0.2 M Bis-Tris buffer pH 5.0, and 1.6 – 2.0 M ammonium sulfate
With Phosphate ION
Large tetragonal crystals were grown in sitting drops at room temperature using reservoir solutions of 0.05– 0.20 M Bis-Tris pH 5.0 – 6.5 and 17 – 25 % PEG 3350. The Pi complex was formed by soaking a crystal for 30 minutes in 21 % (w/v) PEG 3350, 0.1 M Bis-Tris pH 6.0, 25% (v/v) PEG 200, and 20 mM Pi (from a stock solution of 100 mM KH2PO4 adjusted to pH 6.0). The crystal was then picked up with a Hampton loop and plunged into liquid nitrogen.
Target: Histindine Acid Phosphotase
NCBI GENE # or RefSeq#: 5523568
EC #: 3.1.3.2
Length of Protein:342 residues
Molecular Weight:
Possible Items to Do for this project: -- Dr. B
IC50 determination of top compounds
DSF analysis of ligands (machine in Robertus lab)
DSF_NatProt2007.pdf
pharmacophore searching in ICM
http://www.molsoft.com/gui/cheminformatics-tutorials.html#tutorial-3D-pharmacophore
Ensemble docking in ICM and GOLD
-- i.e. docking against multiple receptor conformations. How many PDB structures are there for your enzyme?
In silico alteration of one amino acid residue that is most likely to be mutated in a resistant form of the bacterium and then virtual screening against that structure with your same top hits
-- this mutant version could then be grown up and test in wet lab against the same compounds
Molecular dynamics simulations and re-scoring of top ligands
crystallization
Crystallization Info:
Links: Original Article
PDB Article
WITH L(+)-tartrate ion
Temperature: 293K
Final Optimized condition: 10%(v/v) Tascimate, 0.1 M HEPES pH 7.0 and 19%(w/v) PEG 3350
Extra Info:
Equal volumes of the protein (1.5 ml) and the reservoir
(1.5 ml) were mixed and allowed to equilibrate with 1.0 ml of reservoir
for 24–48 h.
NOTE: Tacsimate (Hampton Research, HR2-755) is a mixture of organic acids that includes sodium malonate, sodium acetate, triammonium citrate, succinic acid, dl-malic acid and sodium formate
Inhibitor Free:
These crystals were grown in sitting drops at room temperature using a protein stock solution of 10 mg/mL enzyme in 50 mM sodium acetate pH 6.0, and reservoir solutions consisting of 0.05 – 0.2 M Bis-Tris buffer pH 5.0, and 1.6 – 2.0 M ammonium sulfate
With Phosphate ION
Large tetragonal crystals were grown in sitting drops at room temperature using reservoir solutions of 0.05– 0.20 M Bis-Tris pH 5.0 – 6.5 and 17 – 25 % PEG 3350. The Pi complex was formed by soaking a crystal for 30 minutes in 21 % (w/v) PEG 3350, 0.1 M Bis-Tris pH 6.0, 25% (v/v) PEG 200, and 20 mM Pi (from a stock solution of 100 mM KH2PO4 adjusted to pH 6.0). The crystal was then picked up with a Hampton loop and plunged into liquid nitrogen.
Forward Primer:
TACTTCCAATCCATGAAGAAAATCTTTGTTAG
Reverse Primer:
TATCCACCTTTACTGTTATTTCTGGAATTTTTCG
Background/Disease:
Image:
CDS GENE SEQUENCE:
>gi|89255449:32306-33361 Francisella tularensis subsp. holarctica LVS chromosome, complete genome
ATGAAAAAAATTTTTGTGTCATTTACATTATTGTTTTTTTTGATACCTGTAGGCTACTCAAGTAAGCTGATTT
TTGTATCGATGATAACACGTCATGGTGATAGAGCACCTTTTGCAAATATCGAAAATGCTAATTATAGCTGGG
GAACTGAGCTATCTGAGCTTACCCCTATTGGGATGAATCAAGAATATAATTTAGGACTTCAGCTAAGAAAAA
GATATATAGATAAATTTGGTCTTTTACCAGAGCATTATGTTGATCAAAGTATCTATGTGCTATCAAGTCACACA
AATCGTACAGTGGTTAGTGCTCAAAGTTTGCTTATGGGTCTATATCCAGCTGGTACAGGACCGCTAATTGG
TGATGGTGATCCAGCAATTAAAGATAGATTTCAACCAATACCAATAATGACCTTGTCAGCAGATTCTCGACT
TATACAGTTTCCTTATGAACAATATTTAGCTGTATTAAAAAAATATGTCTATAACTCACCAGAGTGGCAAAACA
AGACAAAAGAAGCTGCACCAAATTTTGCAAAATGGCAGCAAATACTAGGTAATAGAATCTCAGGATTAAATGA
TGTTATTACAGTAGGAGATGTTTTGATTGTTGCTCAAGCACATGGTAAACCTTTACCAAAGGGTTTATCTCAA
GAAGATGCTGATCAGATAATTGCTCTTACAGATTGGGGACTAGCGCAGCAGTTTAAATCACAAAAAGTATCC
TATATCATGGGTGGTAAATTGACTAATAGAATGATTGAAGATCTAAATAATGCCGTTAATGGTAAATCTAAGTAT
AAAATGACTTACTATTCAGGGCATGATCTGACACTTTTAGAAGTTATGGGTACTTTAGGCGTACCACTAGATA
CAGCGCCAGGATATGCAAGTAATCTAGAAATGGAACTTTATAAAGATGGCGATATCTATACGGTTAAGCTTAG
ATACAATGGTAAGTATGTCAAACTACCTATAATGGATAAAAATAATAGTTGCAGTTTAGATGCTCTAAATAAGTAT
ATGCAGAGTATTAATGAGAAGTTTCAAAAATAA
AMINO ACID SEQUENCE:
>gi|89255478|ref|YP_512839.1| acid phosphatase [Francisella tularensis subsp. holarctica LVS]
MKKIFVSFTLLFFLIPVGYSSKLIFVSMITRHGDRAPFANIENANYSWGTELSELTPIGMNQEYNLGLQLRKRYIDKFGLLPEHYVDQSIYVLSSHTNRTVVSAQSLLMGLYPAGTGPLIGDGDPAIKDRFQPIPIMTLSADSRLIQFPYEQYLAVLKKYVYNSPEWQNKTKEAAPNFAKWQQILGNRISGLNDVITVGDVLIVAQAHGKPLPKGLSQEDADQIIALTDWGLAQQFKSQKVSYIMGGKLTNRMIEDLNNAVNGKSKYKMTYYSGHDLTLLEVMGTLGVPLDTAPGYASNLEMELYKDGDIYTVKLRYNGKYVKLPIMDKNNSCSLDALNKYMQSINEKFQK
NIAID Video
http://www.youtube.com/watch?v=itZE_y5sCFc&feature=youtu.be